P755: STUDIES OF THE SPECIFIC TCIRG1 MUTATIONS CAUSING CONGENITAL NEUTROPENIA
Authors: Vahagn Makaryan, Merideth Kelley, Audrey Anna Bolyard, David Dale
Journal: HemaSphere (2023)

Topic: 11. Bone marrow failure syndromes incl. PNH - Biology & Translational Research Background: Severe congenital neutropenia (SCN) is a hereditary blood disorder associated with recurrent infections and leukemic transformation. We previously identified a novel heterozygous missense mutation (R736S) in TCIRG1 causing SCN in a large multigenerational family (PMC4055522) and a statistically significant correlation of lower ANC with rare missense variants in TCIRG1 through population-based, genome screening (PMC5079157). We have now identified two additional TCIRG1 heterozygous variants: R736C and E722D in two unrelated families with congenital neutropenia. Independently, Shinwari et al identified a novel TCIRG1 mutation, V52L, which was correlated with the neutropenic phenotype (PMC9095858). Homozygous mutations of TCIRG1 cause autosomal recessive osteopetrosis (ARO). Genome Aggregation Database (gnomAD) currently has no data about TCIRG1 R736S, R736C or E722D mutations accruing in the healthy population. This is in contrast to heterozygous variants throughout the TCIRG1 gene in healthy individuals. Aims: We aim to characterize hematological abnormalities caused by mutant TCIRG1 expression utilizing patient derived iPSCs. We will characterize the incidence of TCIRG1-associated neutropenia by communicating this new information within the scientific community through identifying more cases/families with this disease. Methods: We used whole exome sequencing (WES) to identify the genetic basis of neutropenia in the family with R736S mutation. The R736C and E722D mutations also were identified by WES of the index case. Blood counts and Sanger sequencing identified their affected relatives. iPSC lines from two affected individuals harboring TCIRG1 R736S mutation and healthy volunteer were generated using standard methodology. S732F benign heterozygous TCIRG1 variant was generated utilizing CRISPR/Cas9 knock-In (KI) gene editing a healthy volunteer iPSC line. iPSCs hematopoietic differentiation was achieved by using STEMdiff Hematopoietic Kit. Resultant CD34+ cells were differentiated towards neutrophils using a published protocol (PMC9240714). Cell proliferation, survival and differentiation characteristics were measured by automated cell counter and flow cytometry, cell viability staining and surface markers analysis for granulocytic differentiation. Results: The discovery of these families came through the work of the Severe Congenital Neutropenia International Registry and the expanding use of WES to identify the causes of hereditary disorders. With the identification of the index cases, we have identified several affected family members in each family. In all three families the severity of neutropenia is variable and correlates with the incidence of recurrent bacterial infections. Myeloid differentiation of CD34+ cells derived from iPSCs of two patients with R736S mutations revealed maturation, survival and proliferation abnormalities compared to volunteer cells and cell line harboring benign S732F heterozygous TCIRG1 mutation. Patient cells had a 2-fold drop in cell proliferation and cell viability drop by over 70% compared to the benign S732F cell line or normal control. The myeloid differentiation in the patients’ cells, measured by flow cytometry and assessed by CD66b+/CD14+ and CD11b+/CD15+ markers was impaired by 38% and 23% respectively. Summary/Conclusion: The pathogenesis of TCIRG1-associated neutropenia is presently unknown. Our preliminary studies show that patient iPSC derived hematopoietic cells closely recapitulate the neutropenic phenotype. Population based genomic studies; careful family histories and whole exome sequencing will identify new patients with this disorder. Keywords: Neutropenia, Severe congenital neutropenia, Neutrophil, Hematopoietic stem and progenitor cells